In parallel with the Jennerian and modified Jennerian approach to vaccination against rotavirus diarrhea in which an oral live attenuated rotavirus vaccine is generated, we are pursuing other exploratory approaches to vaccination. A complete rotavirus particle consists of three capsid layers: an outer layer consisting of VP4 and VP7, an intermediate layer consisting of VP6 and an inner core layer consisting primarily of VP2. In this project, taking advantage of (i) the availability in our laboratory of various baculovirus recombinants expressing rotavirus proteins including VP2, VP4, VP6 or VP7 and (ii) properties of selected rotavirus structural proteins to self-assemble spontaneously and form virus-like particles (VLPs) upon co-expression in insect cells, we are pursuing the construction of recombinant rotavirus VLP subunit vaccine candidates that can be delivered via various routes, including intranasal. In addition, we are pursuing the construction of recombinant rotavirus subunit vaccines produced from prokaryotic or eucaryotic expression vectors. Such purified protein subunit vaccines conjugated with selected bacterial polysaccharides can be delivered parenterally. Last year, we constructed (i) a baculovirus system expressing the VP8 subunit of VP4 of human rotavirus P serotype 1A (strain Wa). In collaboration with Dr. Szu of LDMI, NICHD, we purified the Wa VP8 protein from insect cells infected with the recombinant baculovirus and immunized mice parenterally with the VP8 protein. Preliminary results showed that the immunized mice developed modest levels of neutralizing antibodies to both homologous Wa virus and to heterologous DS-1 virus. This year, we constructed an E. coli system expressing the VP8 subunit of VP4 of human rotavirus P serotype 1A (strain Wa). Purified VP8 protein induced neutralizing antibodies to both homologous Wa strain and heterologous DS-1 strain upon parenteral immunization in mice. Next, we generated large scale production of the Wa VP8 protein in E. coli.